Capillary electrophoresis is a technique of considerable interest in the analysis of biological mixtures, particularly mixtures of small peptides, proteins and nucleic acids, since it can be used on extremely small samples and permits the use of high voltages which produce separations at high speed. Capillaries also offer the advantage of permitting on-line (i.e., on-tube) detection, a simple and highly efficient means of detection which avoids the need for time-consuming steps such as staining and derivatization of the solutes, and avoids dilution of the solutes and the risk of inaccuracies due to peak broadening or mixing upon emergence of the solutes from the capillary. A particularly useful form of capillary electrophoresis for certain types of biological mixtures, particularly proteins, is isoelectric focusing, which separates solutes on the basis of their isoelectric points.
On-tube detection is an important advantage of capillary electrophoresis since it allows monitoring the separated components while the experiment is in progress without loss in resolution and without the need for staining or derivatizing separated components. However, in capillary isoelectric focusing the components remain focused in stationary zones at the completion of the separation process. Detection of the separated components can be achieved either by scanning the length of the capillary or by mobilizing the focused zones past a single detection point in the capillary (i.e., on-tube detection) or to an external detector. Mobilization techniques are preferred over scanning since they avoid the need for motorized scanning equipment and provide higher sensitivity. Mobilization also permits recovery of isolated zones individually for preparative purposes as they emerge from the capillary.
Mobilization has been achieved in a variety of ways. One method is by hydrodynamic flow, which involves pumping a solution through the capillary to displace the separation medium past the detection point or out of the capillary, depending on which type of detection technique is used and whether or not solute recovery is sought. A problem encountered in this technique is parabolic zone distortion. In certain systems, this can be suppressed by the use of a sucrose gradient in the focusing stage. Unfortunately, such systems are limited to those with a relatively large diameter column, i.e., 3-20mm for example, rather than a capillary, and only when such columns are mounted in a vertical position. A further disadvantage is that hydrodynamic flow mobilization is not possible when the focusing medium is a gel.
An alternative mobilization technique is disclosed by Hjerten, et al., U.S. Pat. No. 4,725,343, issued Feb. 16, 1988. Mobilization according to this technique is achieved by first changing the pH of either the anolyte or the catholyte after focusing, to place the pH's of the anolyte and catholyte either both above or both below the entire range of isoelectric points of the focused ampholyte zones. When a voltage is then applied between the anolyte and catholyte, the entire ampholyte zone pattern moves as a unit through the separation medium. The preferred embodiment in the patent is the replacement of the anolyte used in the focusing step with the same solution used for the catholyte, or vice versa, with the result that the two ends of the separation medium are in electrical contact with identical solutions.
Another alternative is that disclosed by Hjerten, U.S. Pat. No. 4,911,808, issued Mar. 27, 1990. Mobilization according to this patent is achieved by the addition of cations other than protons (such as Na.sup.+, for example) to the anolyte, or anions other than hydroxyl groups (such as Cl.sup.-, for example) to the catholyte. When voltage is applied, migration of these ions into the capillary causes a change in the concentration of the proton or hydroxyl ion, respectively, in the separation medium. The resultant shift in pH imparts charge to the focused proteins and ampholytes, which now migrate in the appropriate direction and are thus mobilized.
These latter two methods are effective for many systems. In some systems, however, these methods do not produce mobilization with equal effectiveness for all peaks. In some cases, for example, late-migrating or slow-moving peaks are broadened during the mobilization, and may not appear at the detection point at all.